Integator system and method for rapidly determining effectiveness of a germicidal treatment

ABSTRACT

The effectiveness of an oxidative sterilization process is determined by exposing a substrate having a known amount of a primary amine or aldehyde indicator chemical to an oxidative germicide. The oxidative germicide reacts with the indicator chemical. The amount of indicator chemical remaining after exposure to the germicide is determined by reacting the indicator chemical with a dye precursor chemical to form a colored product. The amount of indicator chemical remaining on the substrate is determined from the intensity of the color of the colored product. The amount of indicator chemical remaining on the substrate is a measure of the effectiveness of the germicidal treatment. The dye precursor is an aldehyde when the indicator chemical is a primary amine and a primary amine when the indicator chemical is an aldehyde. An integrator for determining the effectiveness of the germicidal process includes a substrate and an indicator chemical, where the indicator chemical is a primary amine or an aldehyde.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an integrator system and a method for rapidlydetermining the effectiveness of a germicidal process for medicalequipment.

2. Background

Medical devices are sterilized before use in hospitals, physicians'offices, and other medical facilities. Steam, heat, ethylene oxide, andhydrogen peroxide are commonly used as sterilizing agents.

It is standard practice to include a sterility indicator in a load ofarticles to be sterilized in a sterilizer. The sterility indicatorprovides a measure of whether the sterilization process was effective insterilizing the articles in a particular load. If the sterilizationprocess was not effective, as indicated by the sterility indicator, theload of equipment is rejected for use.

Biological indicators are generally recognized as reliable sterilityindicators. The biological indicator includes a carrier that has beeninoculated with spores or other microorganisms. Spores are generallyutilized in biological indicators, because spores are more resistant tosterilization than other microorganisms.

The biological indicator is placed into the sterilizer with theequipment to be sterilized. At the end of the sterilization process, thebiological indicator is removed from the sterilizer, and the carrier isimmersed in a sterile culture medium. The culture medium and carrier areincubated for a predetermined time at an appropriate temperature. At theend of the incubation period, it is determined whether anymicroorganisms have grown in the growth medium. If there is no growth ofmicroorganisms in the growth medium, it is assumed that the equipment inthe sterilizer has been properly sterilized. If microorganism growth isobserved, the sterilization process was not effective, and the articlesin the sterilizer are rejected for use. The growth of microorganisms isdetermined through a signal such as the generation of turbidity or acolor change in a pH indicator due to a pH change from byproducts ofcell growth in the medium. Biological indicators are described, forexample, in U.S. Pat. Nos. 5,552,320 and 6,436,659, both of which areincorporated herein by reference in their entirety.

Although biological indicators are accurate indicators for theeffectiveness of the sterilization cycle, at least 24-48 hours arerequired to obtain results from the biological indicators. The equipmentthat was exposed to the sterilization procedure is normally kept inquarantine until the results from the biological indicator areavailable. Medical equipment is expensive, and storage space in medicalfacilities is limited. Some hospitals therefore use the equipment beforethe results are available. Storing quarantined medical equipment is notan efficient use of resources. There is a need for a rapid test fordetermining the effectiveness of the sterilization process.

Foltz et al. (U.S. Pat. No. 6,355,448) describe a method of determiningthe effectiveness of a sterilization process by using the activity ofenzymes rather than spores. It is stated that the enzyme test procedurerequires only a few minutes rather than the several days that arerequired to obtain the results from biological indicators.

The use of a plurality of enzymes rather than a single enzyme wasdisclosed in U.S. Pat. Nos. 5,486,459 and 6,528,277. Using a pluralityof enzymes was believed to better mimic the response of a microorganismthan a single enzyme.

There is a need for sterilization indicators that provide sterilizationresults rapidly.

SUMMARY OF THE INVENTION

One aspect of the invention involves a method for rapidly determiningthe effectiveness of an oxidative germicidal process. The methodincludes providing a substrate having a known amount of a first chemicalon the substrate, where the first chemical is selected from the groupconsisting of a primary amine, mixtures of primary amines, an aldehyde,and mixtures of aldehydes. The first chemical has a first color. Themethod also includes exposing the substrate and the first chemical to anoxidative germicide, thereby decreasing the known amount of the firstchemical to a final amount. The substrate having the final amount of thefirst chemical having the first color is contacted with a secondchemical having a second color, thereby generating a third chemicalhaving a third color. The intensity of the third color is related to thefinal amount of the first chemical on the substrate. The second chemicalis a chemical selected from the group consisting of a primary amine andmixtures of primary amines when the first chemical is a chemicalselected from the group consisting of an aldehyde and a mixture ofaldehydes. The second chemical is a chemical selected from the groupconsisting of an aldehyde and a mixture of aldehydes when the firstchemical is a chemical selected from the group consisting of a primaryamine and a mixture of primary amines. The method also includesdetermining the intensity of the third color and determining theeffectiveness of the germicidal process from the intensity of the thirdcolor.

Advantageously, the effectiveness of the germicidal process isdetermined by correlating the intensity of the third color with resultsfrom biological indicators. In an embodiment, the oxidative germicide isa sterilant. In an alternative embodiment, the oxidative germicide is adisinfectant. The substrate may be an absorbent substrate. Preferably,the substrate is a nonabsorbent substrate.

In an embodiment, the oxidative germicide is a liquid, a vapor, or agas. Advantageously, the intensity of the third color is determinedvisually. Preferably, the intensity of said third color is determinedspectrophotometrically in the visible or ultraviolet region.

In an embodiment, at least one of the first chemical or the secondchemical is colorless. Advantageously, the oxidative germicide isselected from the group consisting of hydrogen peroxide, peracetic acid,ethylene oxide, ozone, and chlorine dioxide. Preferably, the method alsoincludes exposing the substrate and the oxidative germicide to plasma.In an embodiment, the percent completeness of the germicidal process isdetermined by comparing the intensity of the third color with theintensity of the color of a standard. Preferably, the primary amine isglycine or histidine, and the aldehyde is ortho-phthalaldehyde orglutaldehyde.

Another aspect of the invention involves an integrator for determiningthe effectiveness of a germicidal process with an oxidative germicide.The integrator includes a substrate with a known amount of a firstchemical on the substrate, where the first chemical is selected from thegroup consisting of a primary amine, mixtures of primary amines, analdehyde, and mixtures of aldehydes. The substrate is in an enclosure.The first chemical is capable of reacting with the oxidative germicidewhen exposed to the oxidative germicide. The integrator also includes areservoir of a second chemical, where the second chemical is a chemicalselected from the group consisting of a primary amine and mixtures ofprimary amines when the first chemical is a chemical selected from thegroup consisting of an aldehyde and a mixture of aldehydes, and thesecond chemical is a chemical selected from the group consisting of analdehyde and a mixture of aldehydes when the first chemical is achemical selected from the group consisting of a primary amine and amixture of primary amines. The second chemical is capable of reactingwith the first chemical to form a third chemical having a color. Thereservoir has a breakable barrier that isolates the second chemical fromthe first chemical and from the oxidative germicide during thecontacting of the first chemical with the oxidative germicide. Breakingthe breakable barrier in the reservoir contacts the second chemical withthe first chemical, thereby forming the third chemical having the color.The reservoir is in the enclosure.

In an embodiment, the breakable barrier in the reservoir includes afrangible ampoule in the enclosure. Advantageously, the integrator alsoincludes a second barrier, where the second barrier is inside theenclosure between the frangible ampoule and the first chemical. Thesecond barrier in the enclosure is permeable to the second chemical. Thesecond barrier prevents fragments from the frangible ampoule fromcontacting the first chemical.

In an embodiment, the integrator also includes a window in theenclosure, where the window is permeable to the oxidative germicide. Thewindow allows the oxidative germicide to enter the enclosure.Advantageously, the primary amine is selected from the group consistingof glycine and histidine, and the aldehyde is selected from the groupconsisting of ortho-phthalaldehyde and glutaldehyde.

Preferably, the enclosure on the integrator also includes a transparentwindow, where the color change on the substrate can be observed throughthe transparent window visually or with a spectrophotometer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of an integrator according to anembodiment of the present invention;

FIG. 2 is a schematic diagram of a compressible integrator systemcontaining an integrator according to an embodiment of the presentinvention;

FIG. 3 is a schematic diagram of a slidable integrator system containingan integrator according to an embodiment of the present invention; and

FIG. 4 is a schematic diagram of the slidable integrator system of FIG.3 after an outer shell has been moved over an inner shell of a closablesliding container on the slidable integrator system.

DETAILED DESCRIPTION OF THE INVENTION

The term germicide as used herein is meant to include both sterilantsand disinfectants. The term germicidal process as used herein includesboth sterilization processes and disinfection processes. Sterilizationindicators that utilize chemicals to mimic the resistance of abiological indicator (BI) have been termed as integrators. Integratorsutilize an indicator chemical that responds to the germicide that isused in the germicidal process. The chemical reacts with the germicidein a repeatable manner and responds to the factors that are important tosterilization or disinfection in the germicidal process. The reaction ofthe indicator chemical with the germicide is integrated over time, andthe amount of indicator chemical remaining on the integrator iscorrelated with the BI response.

Integrators integrate the reaction of the chemical over time in responseto the critical parameters over a specified range of sterilizationcycles.

The integrators and the method according to embodiments of the presentinvention provide results quickly, reproducibly, and accurately. Thechemicals that are used in the integrators are inexpensive and stable.The results that are obtained from the integrators and the methodaccording to embodiments of the present invention correlate well withthe results from biological indicator tests.

The integrator according to embodiments of the present invention ismeant to mimic the resistance of a biological indicator (BI) withoutusing spores. The integrator according to an embodiment of the presentinvention includes an indicator chemical that reacts with an oxidativegermicide. The integrator is suitable for oxidative germicides includinghydrogen peroxide, peracetic acid, ethylene oxide, ozone, and chlorinedioxide. The oxidative germicides may be in the form of a liquid, avapor, or a gas.

In an embodiment, plasma may be utilized in combination with theoxidative germicides to enhance the reaction of the oxidative germicideswith the microorganisms in the chamber and the indicator chemical on theintegrator and/or to break down the oxidative germicide after use. Theuse of plasma is optional.

The results from the integrator according to embodiments of the presentinvention are available quickly, approximately 30 seconds toapproximately 5-6 minutes, depending on the chemicals selected for theintegrators, compared to the 24-48 hours that are normally required toobtain results from a biological indicator.

Although described in the context of sterilization with a combination ofhydrogen peroxide and plasma with the STERRAD® process, commerciallyavailable from Advanced Sterilization Products of Irvine, Calif., theintegrator according to embodiments of the present invention may be usedwith a variety of germicidal processes. The description of germicidalprocesses such as sterilization or disinfection with hydrogen peroxideand plasma through the STERRAD® process is illustrative only and is notmeant to be limiting.

The integrator according to embodiments of the present inventioncontains an indicator chemical. The indicator chemical reacts with thegermicide and responds to the factors that are important forsterilization. The amount of indicator chemical that remains on theintegrator after exposure to the germicide can be correlated with theresponse of BI's that are placed into the sterilization chamber togetherwith the integrator. The response of a BI is a generally acceptedmeasure of the effectiveness of a germicidal process. The response of aBI in the sterilization chamber can be correlated with response of theintegrators to “calibrate” the response of the integrators with theresponse of a biological indicator.

In an embodiment, primary amines or aldehydes are used as indicatorchemicals in integrators according to embodiments of the presentinvention. Oxidative germicides react with both primary amines andaldehydes. Both primary amines and aldehydes are suitable indicatorchemicals for integrators according to embodiments of the presentinvention.

The amount of the primary amine indicator chemical or the aldehydeindicator chemical that remains on the integrator after the germicideprocess can be used to determine the effectiveness of the sterilizationor disinfection process for the load that is treated in the sterilizer.

The amount of primary amine indicator chemical or aldehyde indicatorchemical that remains on the integrator can be measured in a variety ofways such as instrumental methods, chemical analysis, etc. Any suitablemethod of measuring the concentration of the primary amine indicatorchemical or the aldehyde indicator chemical is suitable.

In an embodiment, the completeness of the germicidal process may beconveniently determined by observing a change in color in theintegrator.

Many primary amines react with aldehydes to form colored products. Theamount of primary amine indicator chemical or the amount of aldehydeindicator chemical remaining on the integrator after exposure to theoxidative germicide can be determined from the intensity of the color ofthe product of the reaction of an aldehyde with a primary amine.

As used herein, an aldehyde that is contacted with a primary amineindicator chemical or a primary amine that is contacted with an aldehydeindicator chemical is termed a “dye precursor”, because the product ofthe reaction of a primary amine with an aldehyde is colored, a “dye”,even if neither the primary amine nor the aldehyde has a color.

The primary amine and the aldehyde can change roles, depending on whichchemical is utilized as the indicator chemical in the integrator. In anembodiment in which a primary amine is the indicator chemical, the dyeprecursor is an aldehyde. In an embodiment in which an aldehyde is theindicator chemical, the dye precursor is a primary amine.

The intensity of the color of the colored product resulting from thereaction of the primary amine with the aldehyde can be used to determinethe effectiveness of the treatment of the load with the oxidativegermicide.

In an embodiment, an integrator according to an embodiment of thepresent invention contains a first chemical having a first color, wherethe first chemical is an indicator chemical. The indicator chemical isselected from the group consisting of a primary amine, a mixture ofprimary amines, an aldehyde, and a mixture of aldehydes.

The integrator containing the indicator chemical is placed into asterilizer with a load of equipment that is to be treated. The load andintegrator are contacted with an oxidative germicide in a sterilizer.The oxidative germicide reacts with the indicator chemical, decreasingthe amount of indicator chemical remaining on the integrator. The amountof indicator chemical remaining on the integrator after the contactingwith the oxidative germicide is a measure of the effectiveness of thegermicidal treatment with the oxidative germicide.

At a point when the effectiveness of the treatment with the oxidativegermicide is to be determined, the integrator containing the firstchemical having the first color is contacted with a second chemicalhaving a second color. The first chemical having the first color is theindicator chemical. The second chemical having the second color is thedye precursor. The dye precursor is a chemical selected from the groupconsisting of a primary amine, a mixture of primary amines, an aldehyde,and a mixture of aldehydes. Primary amines may not be mixed withaldehydes to form the dye precursor. In an embodiment where the firstchemical, the indicator chemical, is a primary amine or a mixture ofprimary amines, the second chemical, the dye precursor, is an aldehydeor a mixture of aldehydes. In an embodiment where the first chemical,the indicator chemical, is an aldehyde or a mixture of aldehydes, thesecond chemical, the dye precursor, is a primary amine or a mixture ofprimary amines.

The product from the reaction of the first chemical, the indicatorchemical, with the second chemical, the dye precursor, is a thirdchemical having a third color. The intensity of the third color of thethird chemical resulting from the reaction of the first chemical withthe second chemical can be used to determine how much of the firstchemical, the indicator chemical, remains on the integrator. The amountof the first chemical indicator chemical that remains on the integratoris a measure of how effective the treatment with the oxidative germicidewas. If only a small amount of the first chemical indicator chemicalremains on the integrator, the intensity of the third color due to thethird chemical is low. A low intensity of the third color is anindication that the treatment with the oxidative germicide waseffective.

In an embodiment, the degree of completeness of the treatment with theoxidative germicide can be determined from the intensity of the thirdcolor due to the third chemical, the product of the reaction of thefirst chemical indicator compound with the second chemical dyeprecursor. The indicator compound on the integrator, the first compound,reacts with the oxidative germicide in parallel with the germicidaltreatment of the load in the sterilization chamber. The intensity of thethird color due to the third chemical resulting from the reaction of thefirst chemical indicator chemical with the second chemical dye precursordecreases as the amount of the indicator chemical decreases due to thereaction with the oxidative germicide.

The intensity of the third color can be correlated with results frombiological indicators that are placed into the sterilization chambertogether with the indicators. The intensity of the third color can becorrelated with the percent sterilization or percent disinfection, asdetermined from biological indicators. The percent sterilization ordisinfection can therefore be determined from the intensity of the thirdcolor due to the third compound.

In an embodiment, the first chemical, the indicator chemical, is placedon a substrate for ease of handling. The substrate can be a variety ofmaterials. The substrate can be an absorbing substrate or a nonabsorbingsubstrate. Absorbing substrates absorb the germicide. Nonabsorbingsubstrates absorb little or none of the germicide.

Filter paper is an absorbing substrate, because filter paper absorbs thegermicide. A glass filter disk is a nonabsorbing substrate, because theglass filter disk does not absorb significant quantities of thegermicide and is thus preferred.

The indicator chemical can be packaged in a water-soluble binder such asan acrylic polymer or carboxymethylcellulose. The indicator chemical andwater-soluble binder can be applied to the surface of the integrator orsterility indicator by, for example, ink jet printing a solution of theindicator chemical and the water-soluble binder onto the surface of aninert backing material.

Absorbing substrates absorb germicide during the germicidal process.When the second chemical dye precursor is contacted with the absorbingsubstrate, the absorbed germicide on the absorbing substrate can reactwith the dye precursor. Oxidative germicides generally react withprimary amines and aldehydes, the two forms of dye precursor. It istherefore generally advantageous to use excess second chemical primaryamine or aldehyde dye precursor when the substrate is an absorbingsubstrate, because the absorbed germicide reacts with the dye precursorwhen the dye precursor is contacted with the absorbing substrate.

In an embodiment, the integrator containing the indicator chemical isplaced in the sterilizer with the equipment to be sterilized and isexposed to the germicide. The indicator chemical reacts with thegermicide, reducing the initial concentration of the indicator chemicalfrom an initial value to a final value.

After the germicidal process is complete, the integrator containing thefirst chemical indicator chemical is exposed to the second chemical dyeprecursor. If any indicator chemical is still present, contacting theindicator chemical with the dye precursor forms the third compoundhaving the third color. If significant color develops on the integrator,the germicidal cycle is judged to have not been effective.

It is generally preferred that the second chemical dye precursor becontacted with the integrator after the conclusion of the cycle, becausethe dye precursor reacts with the germicide. If the dye precursor iscontacted with the integrator before the conclusion of the cycle, thegermicide will react with the second chemical dye precursor, and it maybe necessary to add dye precursor to provide sufficient dye precursor tocause a color change from the reaction of the second chemical dyeprecursor with the first chemical indicator chemical, forming the thirdchemical having the third color. The second chemical dye precursor istherefore generally contacted with the integrator at the end of thecycle. In an embodiment, the cycle may be a canceled cycle.

In an embodiment, the second chemical is isolated from the germicideuntil the conclusion of the cycle. Isolating the second chemical dyeprecursor from the germicide protects the second chemical from reactingwith the germicide and being destroyed.

The color change from the reaction of the first chemical indicatorchemical with the second chemical dye precursor to form the thirdchemical having the third color can be determined visually. Because avisual change is somewhat subjective, the color change is generallydetermined with an optical detector. The optical detector for the colorchange resulting from the reaction of the first chemical indicatorchemical with the second chemical dye precursor can operate at visibleor ultraviolet wavelengths.

The primary amine may be any suitable primary amine. In an embodiment,the primary amine is an amino acid. In an embodiment, the primary amineis selected from the group consisting of arginine, histidine, andcombinations thereof. Other suitable primary amines include thefollowing amino acids: alanine, proline, amino-caproic acid,phenylalanine, tryptophan, methionine, glycine, serine, cysteine,tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine, andhistidine. Peptides or polypeptides formed from any number or type oramino acids are also suitable primary amines.

Arginine is an exemplary primary amine indicator chemical. Argininegives a strong, rapid color change when exposed to aldehydes. Argininealso reacts rapidly with germicides. Arginine is a water-soluble solidthat is conveniently weighed, dissolved in a solvent, and applied to thesubstrate or other support. Other primary amines can be used in otherembodiments, as will become clear in the description and the Examplesbelow.

Arginine has structure I, below.

The NH₂ groups are primary amine groups. The NH groups are secondaryamine groups. Aldehydes often do not react with secondary amine groups.

The aldehyde may be any aldehyde that reacts with primary amines but notsecondary or tertiary amines to produce a color. Aldehydes such as OPA(ortho-phthalaldehyde), glutaldehyde, and aromatic aldehydes aresuitable. Other aldehydes are also suitable.

FIG. 1 shows a diagram of an integrator system 10 according to anembodiment of the present invention. The integrator system 10 of FIG. 1includes integrator chemistry 14 located on integrator strip 16, wherethe integrator strip 16 is an inert material for supporting theintegrator chemistry 14. The integrator strip 16 is generally made of amaterial that does not react with or adsorb the germicide. Theintegrator chemistry 14 includes a first chemical, the indicatorchemical. The integrator strip 16 is a substrate for the integratorchemistry 14.

Peel off label 18 is optionally located on the integrator strip 16.Information on the sterilization cycle can be written on the peel offlabel 18, and the peel off label 18 with the information on thesterilization cycle can be placed into a sterilization logbook. Chemicalindicator strip 20 contains a chemical that undergoes a color changewhen exposed to the germicide. A color change in chemical indicatorstrip 20 simply shows that the chemical indicator strip 20 has beenexposed to the germicide. The chemical indicator strip 20 is not anindicator of the effectiveness of sterilization but is simply anindicator as to whether the chemical indicator strip 20 has been exposedto germicide.

The color change on the chemical indicator strip 20 shows the operatorthat the integrator system 10 should not be used again. Bordeaux Redchanges color when exposed to hydrogen peroxide. Other dyes can be usedon the chemical indicator strip 20 to indicate exposure to othergermicides. Suitable dyes are described, for example, in U.S. Pat. No.5,942,438, which is incorporated herein by reference in its entirety.

After the cycle, the integrator chemistry 14 portion of the integratorstrip 16 is exposed to the second chemical, the dye precursor. Thesecond chemical dye precursor reacts with the first chemical indicatorchemical on the integrator chemistry 14 to form the third chemicalhaving the third color. The presence of a significant amount of color onthe integrator chemistry 14 on integrator strip 16 after exposure of theintegrator chemistry 14 to the second chemical dye precursor indicatesthat the cycle was not effective.

FIG. 2 shows a compressible integrator system 22. Compressibleintegrator system 22 of FIG. 2 includes integrator chemistry 14 onsubstrate 44 located in container 26. Gas permeable surface 24 allowsthe germicide to enter container 26 and contact the integrator chemistry14.

The dye precursor 28 is contained in reservoir 30. Supports 32 arelocated adjacent to reservoir 30. Reservoir 30 protects the dyeprecursor from being destroyed by reacting with the oxidative germicideduring the germicidal cycle.

After the cycle, compressible integrator system 22 is crushed orsqueezed. Supports 32 pierce the reservoir 30, and the second chemicaldye precursor 28 that was contained in the reservoir 30 contacts theintegrator chemistry 14. The second chemical dye precursor reacts withany first chemical indicator chemical that remains after the cycle. Ifany first chemical indicator chemical remains on the substrate 44, thefirst chemical indicator chemical in the integrator chemistry 14 reactswith the second chemical dye precursor to form the third chemical havingthe third color on the substrate 44, indicating that the germicidalprocess was incomplete. A lack of color on the integrator chemistry 14indicates that the germicidal treatment was successful.

FIG. 3 shows a schematic diagram of a slidable integrator system 34.Slidable integrator system 34 includes closable sliding container 36.The closable sliding container 36 is formed of outer shell 38 and innershell 40. Outer shell 38 slides over inner shell 40. Sliding outer shell38 over inner shell 40 opens window 42 in closable sliding container 36.Window 42 allows germicide to enter the interior of closable slidingcontainer 36. The germicide may be a liquid, a vapor, or a gas.

The closable sliding container 36 contains substrate 44 supportingintegrator chemistry 14. The integrator chemistry 14 includes the firstchemical, the indicator chemical. Substrate 44 is located adjacenttransparent window 46 on inner shell 40. Any color change in thesubstrate 44 can be observed through transparent window 46. Substrate 44is the substrate for the indicator chemistry 14. The indicator chemistry14 includes the first chemical, the indicator chemical. In anembodiment, substrate 44 is a glass filter, a nonabsorbing substrate.

The second chemical, the dye precursor 24, is contained in crushableampoule 48 inside closable sliding container 36. Crushable ampoule 48 ismade of a frangible material such as glass. Crushable ampoule 48protects the second chemical, the dye precursor 24, from being destroyedby the germicide during the germicidal process.

Wedge 50 is attached to an inside of outer shell 38 of the closablesliding container 36. Wedge 50 is a projection on the inside of theouter shell 38. In an embodiment wedge 50 has a sharp edge to aid inpenetrating the crushable ampoule 48. Barrier 52 is located insideclosable sliding container 34 between crushable ampoule 48 and substrate44. Barrier 52 prevents fragments of crushable ampoule 48 frominterfering with the reading of substrate 44. Barrier 52 is permeable tothe second chemical, the dye precursor 24. When crushable ampoule 48 iscrushed, the second chemical, the dye precursor, is released and canflow through barrier 52 to contact the first chemical indicator chemicalon substrate 44. In an embodiment, barrier 52 is a wire screen.

FIG. 4 shows a schematic diagram of the slidable integrator system 34and closable sliding container 36 of FIG. 3 after the conclusion of thecycle. The outer shell 38 of the closable sliding container 36 in FIG. 4has been moved toward the left of FIG. 4 by sliding outer shell 38 overinner shell 40.

Sliding outer shell 38 over inner shell 40 has several effects, as shownin FIG. 4. First, sliding outer shell 38 over inner shell 40 closeswindow 42. Closing window 42 isolates closable sliding container 36,retaining the second chemical dye precursor inside the closable slidingcontainer 36. The second chemical dye precursor can stain the hands ofthe operator. Second, sliding the outer shell 38 over inner shell 40forces wedge 50 into contact with crushable ampoule 48, pushing thecrushable ampoule 48 into contact with barrier 52, crushing thecrushable ampoule 48. Crushing the crushable ampoule 48 releases thesecond chemical, the dye precursor 24. The second chemical dye precursor24 that is released flows though barrier 52 and contacts the substrate44.

If any first chemical indicator chemical remains on the substrate 44when the second chemical dye precursor 24 contacts the substrate 44, thesecond chemical dye precursor 24 reacts with the first chemicalindicator chemical in the integrator chemistry 14 to form the thirdchemical having a third color. The third color is distinctive andreadily distinguished from the first color of the first chemical and thesecond color of the second chemical. Barrier 52 prevents fragments ofthe crushable ampoule 48 from contacting the substrate 44 andinterfering with the determination. The color change on the substrate 44can be observed through transparent window 46.

Method of Using the Integrator

The integrator according to an embodiment of the present invention isplaced into the sterilization chamber with the load that is to besterilized, and the germicidal cycle is run. After the completion of thesterilization cycle, the second chemical dye precursor is contacted withthe integrator. The second chemical, the dye precursor, reacts with anyfirst chemical indicator chemical remaining on the integrator to producethe third chemical having the third color. The color that is produceddepends on the structure of the first chemical indicator chemical andthe second chemical dye precursor. The intensity of the color depends onthe amount of first chemical indicator chemical that remains on theintegrator and on the concentration of the second chemical dyeprecursor.

If color develops on the integrator within a predetermined time period,such as approximately 5-6 minutes after the second chemical dyeprecursor is added to the integrator, the sterilization cycle is judgedto have been ineffective.

The intensity of the color on the integrator may be judged visually bycomparing to a color standard, or the intensity of the color may bemeasured spectrophotometrically in the visible or ultraviolet region.Judging the color intensity visually is more subjective than measuringthe color intensity with an instrument.

The second chemical dye precursor may be contacted with the integratorin various manners. In an embodiment, the second chemical dye precursoris contacted with the integrator manually using a pipette, aneyedropper, or other suitable device.

Manual addition of the second chemical dye precursor is appropriate withan integrator such as the integrator shown in FIG. 1, where there is nomethod of protecting the second chemical dye precursor from beingexposed to and being destroyed by the germicide during the sterilizationcycle.

FIGS. 2 and 3 show embodiments of integrators where the second chemicaldye precursor is present in the integrator during the sterilizationcycle but is protected from exposure to the germicide by being enclosedin a reservoir or a crushable ampoule. Other means of protecting thesecond chemical dye precursor from being exposed to the germicide may beused in other embodiments.

The following examples are meant to be illustrative only and are notmeant to be limiting on the scope.

EXAMPLES Example 1 Results of Tests with an Integrator ContainingArginine as the Indicator Chemical With Varying Injection Volumes ofHydrogen Peroxide

A series of integrators was prepared by contacting paper disks with anaqueous solution of arginine. The integrators were placed in a STERRAD®50 sterilizer with a load of equipment to be sterilized and severalbiological indicators. The paper disks are absorbing substrates.

The sterilizer was evacuated to 0.8 torr. Plasma was produced in thechamber for 15 minutes to condition the load. The sterilizer wasevacuated further to 0.4 torr, and hydrogen peroxide was injected andcontacted with the load, integrators, and biological indicators for 6minutes.

The sterilizer was vented with air for 2 minutes. The sterilizer wasevacuated again to 0.5 torr, and plasma was produced for an additional 2minutes. The plasma power was 400 watts for both plasma exposures.

The paper integrator disks were contacted with 100 μL of a 5% solutionof ortho-phthalaldehyde (OPA) dye precursor after the germicide cycle,and the response of the integrators was measured visually after theintegrators had been contacted with the OPA. The results are shown inTable 1 below. TABLE 1 Integrator and Biological Indicator Test ResultsHydrogen Peroxide Injection Volume Biological μL Indicator ResultsIntegrator Color Intensity 100 100% positive  Dark orange color in 3minutes 250 40% positive  Orange color after 5-6 minutes, a few arecolorless 400 0% positive 10-15% show faint color after 5-6 minutes 5000% positive No color

The cycles with 100 and 250 μL of hydrogen peroxide were ineffective, asshown by the positive biological indicator results. The integratorresults were consistent with the biological indicator results, becausethe integrators for the cycles with 100 and 250 μL of hydrogen peroxidehad significant color within 3-6 minutes after being contacted with theOPA. The color is the result of a reaction between unreacted arginine,the primary amine indicator compound, and OPA, the aldehyde dyeprecursor.

In the sterilization cycle with 400 μL of hydrogen peroxide, 10-15% ofthe integrators developed a faint color after 5-6 minutes. None of thebiological indicators in this run were positive. The faint color thatdeveloped on the integrators after 5-6 minutes is a showing that theintegrators provide a more stringent measure of the degree ofsterilization than the biological indicators.

None of the biological indicators in the run with 500 μL of hydrogenperoxide were positive. None of the integrators had any color. Both thebiological indicator results and the integrator results are consistentin showing that the sterilization cycle with 500 μL of hydrogen peroxidewas effective.

The data from the integrators according to an embodiment of the presentinvention were consistent with the data from the biological indicators.However, the results from the integrators were available in 5-6 minutes,compared to 24-48 hours for the biological indicator results.

Example 2 Integrator Response Measured With a Spectrometer

Non adsorbent glass fiber disks were impregnated with an aqueoussolution of arginine. The glass fiber disks are nonabsorbing substrates.The disks were placed in a STERRAD® 50 sterilizer and processed underthe same conditions as in Example 1 with varying injection volumes ofhydrogen peroxide. The quantities of hydrogen peroxide are shown inTable 2 below.

The glass fiber disks were contacted with 50 μL of a 5% aqueous solutionof OPA dye precursor after the cycle was complete. The intensity oflight absorption of the disks was determined with a TAOS TCS230EVMevaluation module color sensor (Parallax, Rocklin, Calif.) atapproximately 470 nm, approximately 550 nm, and approximately 610 nm(red, green, and blue wavelengths).

Hydrogen peroxide injection volumes of 50, 300, and 1000 μL were used.All of the BI's would be negative with a hydrogen peroxide injection of300 μL in the STERRAD® 50 sterilizer. Table 2 summarizes the colorintensities at the 610 nm (blue) wavelength. The integrator responsesshown in Table 2 are the differences between the initial reading and thefinal reading at 30 seconds after addition of OPA. Large numbers for theintegrator response indicate more color and less effectivesterilization. TABLE 2 Hydrogen Peroxide Injection Volume IntegratorResponse (μL) (Range) for Samples 1000  5-17 300 28-47 50 58-85

Small numbers in the integrator response indicate effectivesterilization. The range of 5-17 in the integrator response for thesamples that were exposed to 1000 μL of hydrogen peroxide is consistentwith effective sterilization. The integrator response range of 28-47 forthe samples that were exposed to 300 μL of hydrogen peroxide isconsistent with effective sterilization.

The integrator response range of 58-85 for the samples that were exposedto 50 μL of hydrogen peroxide indicates significant color, showingineffective sterilization.

The results of Example 2 demonstrate that the results from theintegrators according to embodiments of the present invention can bemeasured effectively with a spectrophotometer rather than visually, asin Example 1.

Example 3 Integrator Tests With Histidine as the Indicator Chemical andOPA as the Dye Precursor

Histidine rather than arginine was used as the primary amine indicatorchemical to form a series of integrators in Example 3. An aqueoussolution of histidine was placed on a series of glass fiber disks toform integrators according to an alternative embodiment of the presentinvention.

The integrators were placed into a standard STERRAD® 50 validation load,and cycles were run as described in Examples 1 and 2.

Cycles were run with 0 μL of hydrogen peroxide and 300 μL of hydrogenperoxide. The integrators were contacted with 50 μL of a 5 volume %solution of OPA at the end of the sterilization process. The intensityof the third color of the third compound on the integrators was measuredwith the TAOS color sensor described in Example 2.

A combination of red, green, and blue (RGB) wavelengths (470, 550, and610 nm) were used to measure the response, because the color of theproduct of histidine and OPA is different than the color of the productof arginine and OPA. A root mean square (RMS) of the absorption wascalculated for all three wavelengths measured by the sensor.

The light absorption results were consistent with the treatment with 0μL of hydrogen peroxide being ineffective at sterilization and thetreatment with 300 μL of hydrogen peroxide being effective atsterilization. The example demonstrates that histidine can be used as aprimary amine indicator compound in place of arginine. A wide variety ofprimary amines can be used as primary amine indicator compounds.

Example 4

Integrator Tests With Histidine and Glutaraldehyde

A series of integrators was prepared with histidine as the indicatorchemical on glass fiber disks as the substrate. The integrators wereprocessed in a STERRAD® 50 sterilizer under the same conditions as inExample 3. Glutaraldehyde rather than OPA was contacted with theprocessed integrators as the aldehyde dye precursor. The results areshown in Table 4 below. TABLE 4 Results From Integrator Tests WithHistidine and Glutaraldehyde Range in the Change in Red WavelengthReading Volume of Hydrogen (Change = Initial Color Reading − ColorPeroxide (μL) Reading at 30 Seconds) 0 11-29 300 0-7

The small change in light absorption from the start time to the finishtime for the integrators that were exposed to 300 μL of hydrogenperoxide demonstrates that the sterilization with 300 μL of hydrogenperoxide was effective.

The large change in light absorption from the start time to the readingat 30 seconds for the integrators that were exposed to a volume of 0 μLof hydrogen peroxide is a showing that the sterilization was noteffective.

The results of Example 4 are a demonstration that glutaraldehyde can beused as an aldehyde dye precursor in place of OPA

Example 5 Integrators With OPA as the Indicator Chemical

A series of integrators are prepared with OPA as the indicator chemical.The indicators are placed into a sterilizer with a load to be sterilizedand a series of biological indicators. The load, biological indicators,and integrators are contacted with 100-500 μL of hydrogen peroxide underthe conditions described in Example 1.

The integrators with OPA as the indicator chemical are contacted with anaqueous solution of arginine as the dye precursor. The results from theintegrators with OPA as an aldehyde indicator chemical and arginine as aprimary amine dye precursor correlate well with the results from thebiological indicators.

The results from Example 5 demonstrate that aldehydes such as OPA can beused as the indicator chemical with primary amines such as arginine asthe dye precursor.

The integrator according to embodiments of the present invention allowsthe effectiveness of the sterilization process to be determined rapidly.

Various modifications and alterations of this invention will be apparentto those skilled in the art without departing from the scope and spiritof this invention. It is to be understood that the invention is notlimited to the embodiments disclosed therein, and that the claims shouldbe interpreted as broadly as the prior art allows.

1. A method for rapidly determining the effectiveness of an oxidativegermicidal process, said method comprising: providing a substrate havinga known amount of a first chemical on the substrate, wherein said firstchemical is selected from the group consisting of a primary amine,mixtures of primary amines, an aldehyde, and mixtures of aldehydes,wherein said first chemical has a first color; exposing the substratehaving the known amount of the first chemical to an oxidative germicide,thereby decreasing the known amount of the first chemical to a finalamount of the first chemical; contacting the substrate having the finalamount of the first chemical having the first color with a secondchemical having a second color, thereby generating a third chemicalhaving a third color, said third color having an intensity, wherein theintensity of said third color is related to the final amount of saidfirst chemical on said substrate, wherein said second chemical is achemical selected from the group consisting of a primary amine andmixtures of primary amines when said first chemical is a chemicalselected from the group consisting of an aldehyde and a mixture ofaldehydes, and said second chemical is a chemical selected from thegroup consisting of an aldehyde and a mixture of aldehydes when saidfirst chemical is a chemical selected from the group consisting of aprimary amine and a mixture of primary amines; determining the intensityof said third color; and determining the effectiveness of the germicidalprocess from the intensity of said third color.
 2. The method of claim1, wherein the step of determining the effectiveness of the germicidalprocess comprises correlating the intensity of said third color withresults from biological indicators.
 3. The method of claim 1, whereinsaid oxidative germicide is a sterilant.
 4. The method of claim 1,wherein said oxidative germicide is a disinfectant.
 5. The method ofclaim 1, wherein said substrate is an absorbent substrate.
 6. The methodof claim 1, wherein said substrate is a nonabsorbent substrate.
 7. Themethod of claim 1, wherein said oxidative germicide is a liquid, avapor, or a gas.
 8. The method of claim 1, wherein the intensity of saidthird color is determined visually.
 9. The method of claim 1, whereinthe intensity of said third color is determined spectrophotometricallyin the visible or ultraviolet region.
 10. The method of claim 1, whereinat least one of said first chemical and said second chemical iscolorless.
 11. The method of claim 1, wherein said oxidative germicideis selected from the group consisting of hydrogen peroxide, peraceticacid, ethylene oxide, ozone, and chlorine dioxide.
 12. The method ofclaim 1, further comprising exposing said substrate and said oxidativegermicide to plasma.
 13. The method of claim 1, wherein a percentcompleteness of the germicidal process is determined by comparing theintensity of the third color with an intensity of a color of a standard.14. The method of claim 1 wherein the primary amine is selected from thegroup consisting of glycine and histidine and the aldehyde is selectedfrom the group consisting of ortho-phthalaldehyde and glutaldehyde. 15.An integrator for determining the effectiveness of a germicidal processwith an oxidative germicide, said integrator comprising: a substratewith a known amount of a first chemical on the substrate, wherein saidfirst chemical is selected from the group consisting of a primary amine,mixtures of primary amines, an aldehyde, and mixtures of aldehydes;wherein said substrate is in a enclosure; wherein said first chemical iscapable of reacting with said oxidative germicide when exposed to saidoxidative germicide; a reservoir of a second chemical, wherein saidsecond chemical is a chemical selected from the group consisting of aprimary amine and mixtures of primary amines when said first chemical isa chemical selected from the group consisting of an aldehyde and amixture of aldehydes, and said second chemical is a chemical selectedfrom the group consisting of an aldehyde and a mixture of aldehydes whensaid first chemical is a chemical selected from the group consisting ofa primary amine and a mixture of primary amines; wherein said secondchemical is capable of reacting with said first chemical to form a thirdchemical having a color; wherein said reservoir has a breakable barrierthat isolates the second chemical from the first chemical and from theoxidative germicide during the contacting of the first chemical with theoxidative germicide, wherein breaking said breakable barrier in thereservoir contacts said second chemical with said first chemical,thereby forming said third chemical having the color; and wherein saidreservoir is in said enclosure.
 16. The integrator of claim 15, whereinsaid breakable barrier in said reservoir comprises a frangible ampoulein said enclosure.
 17. The integrator of claim 16, further comprising asecond barrier, wherein said second barrier is inside said enclosurebetween said frangible ampoule and said first chemical, wherein saidsecond barrier in said enclosure is permeable to said second chemical,and wherein said second barrier prevents fragments from the frangibleampoule from contacting the first chemical.
 18. The integrator of claim15, further comprising a window in said enclosure, wherein said windowis permeable to said oxidative germicide, said window allowing saidoxidative germicide to enter said enclosure.
 19. The integrator of claim15, wherein the primary amine is selected from the group consisting ofglycine and histidine and the aldehyde is selected from the groupconsisting of ortho-phthalaldehyde and glutaldehyde.
 20. The integratorof claim 15, wherein said enclosure further comprises a transparentwindow, wherein a color change on said substrate can be observed throughsaid transparent window visually or with a spectrophotometer.